Natural killer (NK) cells represent an important component of the immune system. They are a group of white blood cells (lymphocytes) that can directly kill virus-infected cells and tumor cells. NK cells are essential for resistance to many pathogens, such as viruses, bacteria, and parasites. NK cells differ from B and T lymphocytes in that they respond much faster to infections. Unlike B and T cells, NK cells do not express antigen-specific receptors, which raises the interesting question of how NK cells can detect infected cells. NK cells exert their function in two ways: by producing cytokines, such as interferon-gamma, and by killing infected cells. NK cells can also kill tumor cells. Despite the importance of NK cells in the innate immune response to many types of pathogens, it is still unclear what receptors and what signal transduction pathways control their activation. NK cell inhibitory receptors that recognize surface molecules called major histocompatibility complex class I (MHC class I) prevent the killing of normal, healthy cells.&#8232;The major goal of this project is to define receptorligand interactions that regulate cytotoxicity (lysis of target cells) and cytokine production by NK cells, to determine the precise contribution of individual receptors to signal transduction in NK cells, and to elucidate the mechanism by which inhibitory receptors block NK cell activation. [unreadable] [unreadable] The functional outcome of NK cell stimulation is determined by an intricate balance of signals from activating and inhibitory receptors. But how these signals are integrated and which molecular checkpoint(s) control this process has remained elusive. We have provided evidence that c-Cbl modulates the balance of Vav1 signaling and thereby controls Vav1-dependent synergistic signals by natural cytotoxicity receptors. NKG2D and 2B4 independently induced Vav1 phosphorylation and their combination led to further enhancement, which provided synergistic signals required for cytotoxicity. Inhibition of Vav1 abrogated the synergistic signals for activation, whereas inhibition of c-Cbl potentiated these signals by lowering the activation threshold of the Vav1-dependent pathway. Furthermore, NKG2D and 2B4 synergy, independently of c-Cbl, was dominantly inhibited by CD94/NKG2A through Vav1 dephosphorylation. Our results suggest that c-Cbl acts as a molecular checkpoint by setting an activation threshold via down-modulation of Vav1. [unreadable] [unreadable] The response of NK cells to contact with other cells is regulated through many different receptorligand interactions. It is not yet clear to what extent the distribution and mobility of ligands, anchored into the plasma membrane of target cells, influence NK cell activation and immune synapse formation. The plasma membrane is not homogenous and includes domains referred to as lipid rafts, which are enriched in glycosphingolipids, sphingomyelins, and cholesterol. Whereas lipid rafts play a role in signaling by lymphocyte receptors, it is unclear how partitioning of the receptor ligands into different membrane domains on the target cell may influence cell activation. To investigate the role of the distribution and mobility of ligands on target cells, and to avoid the use of toxic inhibitors such as cyclodextrin, cholesterol-auxotroph insect cells (Drosophila S2 cells) were grown in serum-free conditions to eliminate cholesterol-enriched domains. We have shown that the GPI-linked CD48 (the ligand for the NK cell receptor 2B4) is associated with the detergent-resistant membrane (DRM) fraction of S2 cells and its association with this fraction is weaker in cholesterol-free S2 cells. In contrast, ULBP1 (the ligand for the NK cell receptor NKG2D) is distributed both in the DRM and soluble fraction of S2 cells and its localization does not change in the absence of cholesterol. 2B4 signaling in freshly isolated, resting NK cells and in IL-2activated NK cells, was reduced when CD48 was expressed in cholesterol-free S2 cells. Surprisingly, NKG2Ddependent activation was increased in IL-2activated NK cells when ULBP1 was expressed in cholesterol-free S2 cells. These data suggest that membrane micro-domains on target cells modulate the response of NK cells.[unreadable] [unreadable] Natural killer (NK) cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR) family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses. [unreadable] [unreadable] NK cell cytotoxicity is achieved by polarized release of perforin-containing granules towards target cells. As polarization and degranulation can be controlled by separate signals, their respective sensitivity to inhibitory receptors and the requirements for inhibition were evaluated. Expression of HLA-C or HLA-E on the human cell line 221 blocked granule polarization, degranulation, and CD16-dependent MIP-1a secretion by NK cell clones with inhibitory receptors of matching HLA specificity. However, HLA-C or HLA-E on Drosophila S2 cells did not fully inhibit CD16-dependent degranulation and MIP-1a secretion, suggesting that other receptorligand interactions, which occur during contact with 221 cells, are required for complete inhibition. In contrast, HLA-C or HLA-E on S2 cells were sufficient to block granule polarization induced by LFA-1 or by NKG2D. Therefore, engagement of inhibitory receptors by HLA class I on target cells is sufficient to block different signals for granule polarization, but not degranulation.[unreadable] [unreadable] Many cellular responses, such as autoimmunity and cytotoxicity, are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIM). We have shown that binding of inhibitory NK cell receptors to HLA class I on target cells induced tyrosine phosphorylation of the adapter Crk, concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore, Crk dissociated from the guanine exchange factor C3G and bound to tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity, providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex, observed here with two types of inhibitory receptors, expands the signaling potential of the large ITIM-receptor family, and reveals an unsuspected component of the inhibitory mechanism.